1. Field of the Invention
This invention relates to the identification of clinical uses of compounds that include L-arginine and various biologically available metalloarginates. In particular, the invention comprises L-arginine, magnesium L-arginate, zinc L-arginate, and copper L-arginate with their ascorbate salts, and their uses as oral nutritional supplements and clinical agents.
2. Description of the Prior Art
(a) L-Arginine and Nitric Oxide (NO)
L-arginine is the precursor amino acid critical to the synthesis of nitric oxide by nitric oxide synthase (cNOS or eNOS), the enzyme responsible for constitutive production of NO by vascular endothelial and neural cells.
Vascular endothelial dysfunction secondary to endothelial stress, ischemia, atherosclerosis, hypertension, aging, diabetes or other etiologies, results in a reduction of the production of NO. Endothelial dysfunction has been well documented in the coronary circulation, and probably also the renal circulation, of patients with essential hypertension and, because of the reduced production of local levels of cNOS, aging alone or aging accompanied by atherosclerosis of the vascular endothelium reduce its ability to produce NO.
NO increases local vascular levels of guanosine 3xe2x80x2,5xe2x80x2-cyclic monophosphate (cGMP), by providing the molecular radical necessary for the production of cGMP via the enzyme guanylate cyclase. Adequate cGMP results in: increased local blood flow, increased smooth muscle relaxation, increases in healthy endothelial cell proliferation, reduced endothelial permeability, inhibition of vascular smooth muscle cell (VSMC) proliferation, inhibition of cellular (both neural and glial) apoptosis and useful alterations in the ciliary muscle and trabecular meshwork of the eye which result in improvements in outflow facility.
There is some evidence that NO alone (also see magnesium, below) modulates intracellular calcium (Ca+2) oscillations by blocking endoplasmic reticular Ca+2 release and by enhancing intracellular Ca+2 extrusion.
NO is not only helpfully antithrombotic, it also strongly counters the potent endothelial-derived vasoconstrictor, endothelin-1 (ET-1). A fine balance of NO and ET-1 not only mediates local blood flow but also many facets of systemic vascular autoregulation. If NO is reduced, an unopposed tonic, ET-1-induced vasoconstrictive response occurs.
Plasma levels of L-arginine can be increased by oral administration. In young men with coronary artery disease, oral L-arginine has been shown to improve endothelium-dependent dilatation and reduce monocyte/endothelial cell adhesion. Dietary L-arginine improves NO-dependent vasodilator function in cholesterol-fed rabbits and blocks the progression of aortic plaques (see below). This apparently occurs via the restoration of cNOS and a reduction of vascular oxidative stress. Plasma arginine concentration is reduced in diabetic rats but is restored to normal following dietary L-arginine supplementation.
Oral doses of L-arginine raise levels of available NO. In one study, chronic administration of L-arginine (2% in drinking water) appeared to improve endothelium-dependent vasodilator function and systemic NO production and to reduce vascular oxidative stress in cholesterol-fed rabbits with pre-existing hypercholesterolemia.
(b) Magnesium (Mg+2)
Mg+2 deficiencies are widespread in the general population. This is especially true in diabetic patients with normal renal function, in the institutionalized elderly, in chronic alcoholics and smokers, and in populations living in regions with water supplies low in Mg+2.
Mg+2 deficiency has been associated with cardiac arrythmias, hypertension, decreased vascular intraluminal fluidity and vasoconstriction. These pathologies result from a reduction in the efficiency of the modulation of cell membrane Ca+2 channel gating. By a variety of mechanisms, Mg+2 modulates the level of both intracellular and extracellular Ca+2. Excess cytoplasmic free Ca+2 has the deleterious effect of leading to an increase in ET-1, with its associated increase in vasoconstriction and cell apoptosis. Mg+2 optimizes cytoplasmic free Ca+2 levels without interfering with normal Ca+2 intracellular signaling. Because vascular endothelial production of ET-1 is highly dependent upon cytosolic Ca+2 influx via transmembrane Ca+2 channels, Ca+2 channel blockade reduces this influx and reduces the production of ET-1.
The eye maintains the equilibrium of intraocular pressure by a dynamic balance between aqueous humor production and aqueous outflow via the trabecular meshwork. If outflow is reduced intraocular pressure rises, and vice versa. Levels of ET-1 are elevated in the aqueous humor of patients with chronic open angle glaucoma, and local production of cNOS is reduced in these eyes. Induced elevations of aqueous ET-1 levels produce optic nerve collapse in rabbits and monkeys. The same critical vasoactive balance between ET-1 and NO appears to be as important in regulating vascular flow within the eye as it is in regulating systemic blood flow. Imbalances that result in uncontested activity of ET-1 because of reduced levels of NO, are strongly implicated in the vascular progression of visual field loss associated with chronic glaucoma.
In addition to these vascular effects, trabecular smooth muscle contraction is produced by ET-1 and strongly opposes the tonic, relaxation properties of NO. As a result, trabecular contraction in the eye is stimulated, resistance to aqueous outflow is increased and IOP increases. At the same time this pressure increase is occurring, putative vasoconstriction of the small vessels of the optic nerve (probably the posterior ciliary arteries) occurs, and the course is set for optic nerve atrophy and blindness.
Clinical confirmation of these findings is found in the fact that exposure of patients to vasodilating stimuli or to Ca+2 channel blockers has resulted in an improvement of the glaucomatous visual field; a serendipitous reduction of IOP has been observed as a side effect in glaucoma patients using Ca+2 channel blockers for systemic hypertension. Unfortunately, pharmaceutical Ca+2 channel blockers used clinically at effective doses have gross, amplitude driven, essentially uncontrolled effects on Ca+2 cellular signaling. As a result, prescribing pharmaceutical doses of blockers to systemically normotensive glaucoma patients subjects the glaucomatous optic nerve to a significant risk of hypoxia secondary to reduced perfusion pressure and leads to an increased risk of visual loss. The sensitive ability of Mg+2 to optimize cytoplasmic free Ca+2 levels permits more delicate control of the modulation of ET-1/NO balances without interfering with normal Ca+2 intracellular signaling and avoids the risks associated with iatrogenic hypotension.
The correction of an existing Mg+2 deficiency exerts counter-apoptotic, antihypertensive, anti-atherosclerotic, anti-arrhythmic, antithrombotic effects and result in a reduction in the risk of visual loss in chronic glaucoma. The prophylactic nutritional supplementation of Mg+2 that prevents the development of magnesium deficiencies, particularly in identifiable groups, will reduce the risks associated with hypomagnesemia and avoid the undesirable side effects associated with powerful Ca+2 channel blockers.
(c) Zinc (Zn+2) and Copper (Cu+2)
Zn+2 and Cu+2 are cofactors for, and are reported to be inducers of, the antioxidant superoxide dismutase (CuZnSOD). Zn+2 also binds the sulfhydryl groups of proteins, additionally protecting them from oxidation.
CuZnSOD enzymatically degrades the free radical superoxide (Oxe2x80xa2). Although Oxe2x80xa2itself is not as damaging to cell membranes as other free radicals, it spawns the highly toxic hydroxyl radical (OHxe2x88x92) via a Fenton reaction with iron. Because constitutively produced NO (from cNOS) is readily inactivated by Oxe2x80xa2, the bioactivity of this endothelium-derived NO is dependent on the local availability and activity of CuZnSOD. In addition, Oxe2x80xa2 interacts with excessive, macrophage-generated (Type II iNOS-derived) NO in inflamed or irritated tissues and generates peroxynitrite, a long-lived and highly reactive oxygen species which is implicated in coronary atherosclerosis. Tyrosine nitration of this peroxynitrite causes endothelial dysfunction by disrupting the integrity of the cell membrane. This peroxynitrite is also implicated in the caspase cascade leading to apoptosis in neurologic conditions. CuZnSOD protects against potentially harmful effects that may be associated with excessive levels of Type II iNOS-generated NO. In addition, there is some evidence that CuZnSOD may alleviate retinal lesions caused by intraocular pressure elevation.
Zn+2-deficient rats develop increased erythrocyte fragility and reduced CuZnSOD activity. They are particularly vulnerable when the metal deficiency is combined with a deficiency of anti-oxidants.
Appropriate dietary Cu+2 is essential for cardiovascular homeostasis. Cytosolic CuZnSOD is inactivated in diets deficient in Cu+2. Studies of tissue microcirculation demonstrate that Cu+2 is important in mechanisms of macromolecular leakage, platelet-endothelial interactions and vascular smooth muscle reactivity. These abnormalities are not surprising in view of the role of CuZnSOD and NO in maintaining vascular integrity. Dietary Cu+2 appears to be necessary for several microvascular control mechanisms affecting the regulation of peripheral blood flow; NO-mediated arteriole vasodilation is compromised in copper-deficiency. Functional deficits of NO can be reversed by the dietary addition of CuZnSOD, suggesting that degradation of NO by Oxe2x80xa2 may occur during Cu+2 deprivation.
Although seemingly not uncommon, dietary deficiencies of these two cofactors, Zn+2 and Cu+2, should be avoided.
(d) Ascorbate (ascorbic acid, vitamin C)
Ascorbic acid (AA) is a powerful antioxidant, a cofactor in collagen synthesis, affects platelet activation and prostaglandin synthesis, and inhibits the polyol (sorbitol) pathway. In diabetics or patients with insulin resistance, net circulating concentrations of AA are reduced because the cellular uptake of ascorbate is promoted by insulin; because the cellular uptake of AA is concurrently reduced by hyperglycemia, there is a secondary, excessive excretion by the diabetic kidney.
AA improves impaired acetylcholine-induced vasodilation by mechanisms linked to NO formation.
AA may mimic glutathione in the stimulation of CuZnSOD. As a substitute, free standing and powerful free radical scavenger, it preserves constitutive levels of glutathione.
AA phase transfer rejuvenation of xcex1-tocopherol maintains and amplifies the latter""s chain-breaking effect on lipid peroxidation, and similarly complements xcex1-lipoic acid; xcex1-tocopherol and xcex1-lipoic acid constitute the ultimate protection from free radical damage to cell membranesxe2x80x94especially those of the vascular endothelium.
Metal L-arginine complexes used in the practice of certain embodiments of this invention have the following formula:
(Arg) M X
in which:
The symbol xe2x80x9cArgxe2x80x9d represents the amino acid L-arginine or bis-L-arginine;
M is either Mg+2, Cu+2 or Zn+2; and
X is either hydroxide, halide, sulfate, phosphate, acetate, ascorbate or bis-ascorbate.
Other embodiments of this invention involve the use of non-metal-containing L-arginate complexes of the formula
(Arg) X
in which:
Arg is either L-arginine or bis-L-arginine; and
X is either hydroxide, halide, sulfate, phosphate, acetate, ascorbate or bis-ascorbate.
In certain aspects, this invention resides in a method for the oral administration of L-arginine ascorbate or metalloarginate complexes, alone or in combination, as a nutrient for humans. The cation of the metalloarginate complexes may be Mg+2, Cu+2 or Zn+2. In other aspects, the invention resides in a method for the oral administration of L-arginine ascorbate, bis-L-arginine ascorbate, or L-arginine bis-ascorbate, either alone or in combination, for the same purposes.
The compound is preferably administered in an oral daily dosage with preferred and most preferred amounts of the individual components as shown below.
Embodiments of the invention include administration in unit dosage forms that include tablets, capsules, powders, suspensions and liquids. The invention contemplates delivery vehicles of, among others, timed release, sustained release, controlled release, or zero-order release in substantially homogeneous or in coated bilayered release unit dosage forms with differential dissolution rates.
The present invention provides for the clinical use of defined arginate complexes which provide concurrent, pharmacologically appropriate dosage forms and amounts of L-arginine, Mg+2, Cu+2, Zn+2 and ascorbate for the beneficial modification of conditions and functions either acutely or chronically associated with dysfunctional vascular endothelium or vasoconstriction, and for various clinical presentations adversely affected by dysfunctional vascular endothelium or vasoconstriction. It is particularly useful in any clinical diagnosis or finding in which it is important to ensure appropriate levels of cNOS, eNOS and NO.